Mark G. P. Saifer

Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD (“relative titer”) had a median of 1.1 (range 0.9–1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1–20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.

Preparation of Long-Acting Superoxide Dismutase Using High Molecular Weight Polyethylene Glycol (41,000-72,000 Daltons)

Superoxide dismutase (SOD) has demonstrated therapeutic potential for treating a variety of conditions including radiation injury, oxygen toxicity, reperfusion injury, and inflammation, especially arthritis. However, the native enzymes short half-life in plasma (6 minutes in mice, 25 minutes in man) limits the enzymes effectiveness in many applications, or requires infusion of large doses. High doses of SOD derived from either natural or rDNA sources may increase the potential for immunologic sensitization. One effective use of native SOD is intra-articular administration for treatment of arthritis, where injection of SOD into joints retards elimination (15 hour terminal half-life), allowing the effective use of lower doses. To overcome the limitations resulting from rapid clearance, various researchers have increased the persistence of SOD by cross-linking SOD or by attaching polymeric substances, including dextrans, albumin, Ficoll, polyvinyl alcohol or polyethylene glycol (PEG). PEG is relatively safe; however, the amount of modification by PEG, is the MW range 1,900-5,000 daltons, which is necessary to optimally increase serum persistence and reduce immunogenicity, results in the loss of much of the enzymatic activity. In this report we describe the preparation of SOD adducts containing 1 to 4 strands of high MW PEG (41,000-72,000 daltons). The MW range of these adducts, measured by steric exclusion HPLC based on protein standards, is 200,000 to over 1,000,000 daltons. The number of PEG strands attached per SOD dimer (32,000 daltons) was measured by HPLC. Because of the low degree of protein modification required to produce very high MW products, these PEG-SODs retain 90%-100% of the SOD activity of the native enzyme. Additionally, these very large adducts demonstrate longer persistence and lower immunogenicity and antigenicity compared to the more highly modified PEG-SODs containing low MW PEG (i.e., 7-16 strands of 5,000 dalton methoxy-PEG).

Plasma Clearance and Immunologic Properties of Long-Acting Superoxide Dismutase Prepared Using 35,000 to 120,000 Dalton Poly-Ethylene Glycol

Some biological properties of bovine and recombinant human Cu,Zn superoxide dismutase (bSOD and rhSOD)-poly-ethylene glycol (PEG) adducts prepared by coupling 1-9 strands of high molecular weight PEG (35,000-120,000 daltons) are compared to SOD adducts coupled with 7 or 15 strands of low molecular weight PEG (5,000 daltons). Plasma clearance after i.v. injection was measured in mice and dogs. Conjugates of bSOD with 2 strands of PEG 40,000, 3 strands of PEG 72,000 or 1 strand of PEG 100,000 demonstrated half-lives of about 36 hours in mice, whereas the half-life of a conjugate with 7 strands of PEG 5,000 was about 24 hours. A PEG-bSOD with an average of 3.3 strands of PEG 41,000 was cleared from plasma with a terminal half-life of 36 to 48 hours after intraperitoneal injection in mice. PEG-SODs prepared from bSOD and rhSOD with 3 strands of PEG 50,000 each had plasma half-lives of approximately five days in dogs. An enzyme immunoassay (ELISA) was employed to measure cross-reactivity with a rabbit antibody directed against bSOD. A series of bSOD adducts with 2 to 9 strands of PEG 35,000-120,000 were compared to PEG-bSODs with 7 or 15 strands of PEG 5,000. Attaching larger PEG strands was at least 3 times more effective in reducing antigenicity, compared to PEG 5,000. Ability to induce sensitizing antibodies was measured using subcutaneous sensitization followed by i.v. or s.c. challenge in mice. Some bSOD conjugates with either 7 or 15 strands of PEG 5,000 induced sensitization reactions before the sixth challenge. Fewer than 1% of the animals tested with bSOD or rhSOD adducts with 3 or 4 strands of PEG 65,000, or 3 strands of PEG in the 30,000-50,000 molecular weight range, showed signs of anaphylaxis during six or seven challenges. In a passive cutaneous anaphylaxis test (optimized to measure mouse IgE), neither bSOD coupled to 2 strands of PEG 120,000 nor bSOD coupled to 9 strands of PEG 35,000 induced detectable antibodies against either of these PEG-bSOD preparations or against bSOD; however, the adduct with 2 strands of PEG 120,000 reacted weakly with pre-formed antibodies to bSOD. The high molecular weight PEG-bSODs tested were not immunogenic, but were weakly antigenic, compared to bSOD.

Pharmacokinetics and tolerability of ventricularly administered superoxide dismutase in monkeys and preliminary clinical observations in familial ALS

The discovery of mutations in the gene for Cu/Zn superoxide dismutase (SOD) in some cases of familial amyotrophic lateral sclerosis (FALS) provides a rationale for enzyme replacement therapy. The inability of SOD to cross the blood-brain barrier motivated this study of the safety, tolerability and pharmacokinetics of bovine SOD (bSOD) administered into the CSF of rhesus monkeys and one late-stage, SOD-deficient FALS patient. Kinetic analyses in the patient indicated that intracerebroventricular (i.c.v.) administration, but not lumbar administration, delivered bSOD to the entire CSF pathway. Daily bolus i.c.v. injections (32 mg/day) and continuous i.c.v. infusion (30 mg/day) were well tolerated by the patient. During the period of daily bolus injections, the patients performance on manual muscle tests was nearly stable, in contrast with the rapid decline before and after that period. These results justify further investigation of bSOD therapy in SOD-deficient FALS patients.